B. Kubanek

The effect of reduced oxygen tension on colony formation of erythropoietic cells in vitro

Summary. The effect of reduced oxygen tension and the role of cellular components known to protect the cell against oxygen toxicity has been studied with respect to erythropoietic colony formation in vitro. Alphathioglycerol can be partially replaced by vitamin E and completely replaced by reduced glutathione (GSH) at physiological concentrations. Incubation of bone marrow and fetal Iiver early (BFU‐E) and late (CFU‐E) erythropoietic progenitor cells, in the presence of GSH, in an atmosphere containing 5% oxygen, 5% carbon dioxide and 90% nitrogen, as opposed to air supplemented with 5% carbon dioxide, resulted in an increase in colony numbers and response to erythropoietin (Epo). The number of colonies derived from bone marrow and fetal liver CFU‐E increased by 1.2‐2.8‐fold with a relative Epo sensitivity increase of 3.5‐4‐fold. Bursts obtained from bone marrow and fetal liver BFU‐E increased from 2.6‐ to 3.8‐fold with an increased response to Epo of 2‐3‐fold. The effects of GSH and low oxygen tension are interpreted as causing a reduction in oxygen toxicity of the cells, thereby increasing the life span in vitro and so increasing the number of cells capable of forming colonies. The heightened response of BFU‐E to Epo, analogous to the effect seen for CFU‐E, implies that BFU‐E may be responsive to physiological Epo concentrations at physiological oxygen tensions.

Mini-pool screening by nucleic acid testing for hepatitis B virus, hepatitis C virus, and HIV: preliminary results

BACKGROUND: The purpose of this study was to evaluate the feasibility of nucleic acid testing (NAT) of mini‐pools as a blood donation screening test.

Granulocytic progenitor cells in aplastic anaemia.

The characteristics and the concentration of granulopoietic colony forming cells (CFC) were examined in 22 patients with aplastic anaemia at different stages of their disease. Additionally the ability of the patients’peripheral leucocytes to elaborate factors necessary for colony stimulation in vitro (CSA) was studied. The ability of the patients’cells to generate CSA was shown to be unaffected. However, the incidence of CFC within the marrow and peripheral blood suspensions was significantly reduced in all patients. The results suggest a reduced compartment size of CFC even in those patients who have recovered from aplastic anaemia. This may indicate that the disturbances in the preceding compartments of the haemopoietic cell renewal system still persist after recovery from the acute bone marrow failure.

A sensitive sandwich ELISA for measuring erythropoietin in human serum

A sandwich, non‐competitive enzyme‐linked immunosorbant assay (ELBA) for erythropoietin (EPO) is described. The ELISA utilizes a monospecific, polyclonal antibody raised in rabbits against human recombinant EPO (rhu EPO) and purified over a rhu EPO affinity chromatography column. The ELISA procedure can be summarized as follows: Anti‐EPO is coated onto 96‐well ELISA microtitre plates; standard EPO or sample is added and left to bind to this catching antibody: this is followed by the addition of the same antibody which has been biotinylated; finally, anti‐biotin conjugated to alkaline phosphatase is added and the enzyme reaction developed and read at 405 nm. All parameters of the assay have been optimized. Recombinant human EPO was standardized against the World Health Organization 2nd International Reference Preparation for erythropoietin. The minimal detectable concentration of rhu EPO was 0.3–0.5 mil/ml, which corresponded to 1.2–2 mU/ml of EPO in serum (serum diluted 1:4). No reaction was obtained with a variety of blood components and cytokines, indicating that the anti‐EPO antibody did not cross‐react with those substances to produce false‐positive results. The intra‐assay variation ranged from 3% to 10%, while the inter‐assay variation ranged from 8.5% to 24%. Serum dose‐response curves were parallel to the standard dose‐response curve. The assay is easy to use, rapid, reproducible, but above all quantitative, specific and sensitive to measure the EPO content in all serum samples.

THE IN VITRO DIFFERENTIATION OF DENSITY SUB‐POPULATIONS OF COLONY‐FORMING CELLS UNDER THE INFLUENCE OF DIFFERENT TYPES OF COLONY‐STIMULATING FACTOR

The in vitro proliferation and differentiation of myeloid progenitor cells (CFU‐c) in agar culture from CBA/Ca mouse bone marrow cells was studied. Density sub‐populations of marrow cells were obtained by equilibrium centrifugation in continuous albumin density gradients. The formation of colonies of granulocytes and/or macrophages was studied under the influence of three types of colony‐stimulating factor (CSF) from mouse lung conditioned medium CSFMLCM), post‐endotoxin mouse serum (CSFES) and from human urine (CSFHu). The effect of the sulphydryl reagent mercaptoethanol on colony development was also examined. The density distribution of CFU‐c was dependent on the type of CSF. Functional heterogeneity was found among CFU‐c with partial discrimination between progenitor cells forming pure granulocytic colonies and those forming pure macro‐phage colonies. Mercaptoethanol increased colony incidence but had no apparent effect on colony morphology or the density distribution of CFU‐c.

Thrombopoietin serum levels in patients with aplastic anaemia: correlation with platelet count and persistent elevation in remission

In an attempt to evaluate the role of thrombopoietin (TPO) in the pathobiology of aplastic anaemia (AA), we have examined TPO levels in sera from 54 AA patients and 119 healthy controls. A total of 92 samples were collected from AA patients: 43 samples were harvested at diagnosis, 23 samples in the cytopenic period after treatment, and 26 samples when patients were in partial (n = 10) or complete remission (n = 16) following immunosuppressive treatment. TPO serum levels were assessed by a sandwich‐antibody ELISA that utilized a polyclonal rabbit antiserum for both capture and signal. Serum samples from normal donors revealed a mean TPO level of 95.3 ± 54.0 pg/ml (standard deviation). Mean TPO levels in AA sera collected at diagnosis and before onset of treatment were 2728 ± 1074 pg/ml (P < 0.001 compared to normal controls; mean platelet count at that time: 27 × 109/l). TPO serum levels of AA patients in partial or complete remission after immunosuppressive treatment were significantly lower than TPO levels at diagnosis (P < 0.001). However, despite normal platelet counts (mean 167 × 109/l), TPO levels remained significantly elevated in complete remission (mean TPO 1009 ± 590 pg/ml, P < 0.001 compared to normal controls). There was a significant inverse correlation between serum TPO levels and platelet counts in AA patients who were not transfused for at least 2 weeks prior to sample collection (coefficient of correlation (r) = −0.70, P < 0.0001).

Serum erythropotietin and serum transferrin receptor levels in aplastic anaemia

Summary. Serum erythropoietin (EPO) and soluble transferrin receptor levels were serially measured in 74 patients with aplastic anaemia (AA). As control groups we investigated healthy controls (n = 24) and patients with iron‐deficiency (n = 23) or haemolytic anaemia (n = 16). There was a significant negative correlation of log EPO on haematocrit both in AA patients and in the anaemic control group. However, for the same degree of anaemia, log EPO levels in AA were significantly higher than in iron‐deficiency or haemolytic anaemia. EPO levels at diagnosis did not correlate with severity of aplastic anaemia, nor did they predict outcome after immunosuppression. During immunosuppressive treatment of AA with anti‐thymocyte globulin and cyclosporine A, EPO levels were significantly lower compared with pre‐treatment values without a corresponding change in haematocrit. This impaired EPO response to anaemia during immunosuppression might affect recovery of erythropoiesis. In AA patients, EPO levels declined with haemopoietic recovery. However, compared with normal controls, EPO levels in remission patients were still higher with respect to their haematocrit.

Pathophysiology of Aplastic Anaemia

Aplastic anaemia is the clinical expression of reduced influx into the compartments of morphologically identifiable proliferating and maturing haemo‐poietic cells from the stem cell compartment. The bulk of evidence, especially the results of bone marrow transplantation, favours a primary alteration of the proliferation and/or differentiation of pluripotent haemopoietic stem cells. Experimental and clinical data suggest that a stem cell compartment which is reduced in size may compensate its output by adaptation of kinetics for a limited period of time; this could explain the well‐known time lag between exposure to potentially harmful drugs or viruses and manifestation of aplastic anaemia. Humoral regulating factors, as far as have been examined, show normal reactivity. A primary alteration of bone marrow stroma is less likely. However, the basic mechanism of aplastic anaemia may be heterogenous and some cases may be due to alteration in the micro‐environment of haemopoietic cells. In many, and maybe in all cases, haemopoietic insufficiency is induced by external factors on the basis of individual hypersensitivity. Chemical, physical and viral factors are known. Direct toxicity of these factors on stem cells is more likely than mediation through immune mechanisms.

Micromegakaryocytes in Human Bone Marrow

Micromegakaryocytes (MMK) were defined morphologically by the cell area, nucleus form and cytoplasmic structure. Bone marrow smears of 7,156 patients were retrospectively analyzed. MMK were found most frequently and abundantly in acute non-lymphatic leukaemia, chronic myeloid leukaemia and pre-leukaemia. The presence of more than 10% MMK in the megakaryocyte population suggest a pre-leukaemic condition or non-lymphatic leukaemia. The platelet production of MMK is probably quantitatively normal although a functional defect is suspected.

In vitro Platelet Function during Storage in Three Different Additive Solutions

Three different synthetic media without glucose were studied for platelet storage. The first medium contained acetate and gluconate. The second contained acetate, gluconate and citrate. Finally the third contained phosphate and mannitol. The purpose of the study was to investigate whether there were differences among the various media in terms of preservation of platelet quality. Pools of platelet concentrates were prepared from buffy coats. In vitro function and metabolic parameters were measured during 5 days of storage in these additive solutions as well as in plasma. Platelet aggregation, hypotonic shock response and release of β‐thromboglobulin, platelet factor 4 and lactate dehydrogenase of the cytosol were equivalent in the media containing acetate compared to plasma storage. In vitro platelet functions and pH in these two media were better preserved compared to the medium with phosphate and mannitol. In addition bacteriological studies using platelets suspended in additive solutions or in plasma were carried out. Carryover of 20% of plasma to the synthetic media necessary for successful platelet storage in these additive solutions allows bacteriological growth. As shown, inoculation of 1 colony/ml Staphylococcus epidermidis leads to 106–107 organisms/ml after 5 days of storage.